Dihydrofolate reductase (DHFR) is a small, ~20 kD enzyme that catalyzes the reduction of 7,8- dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF), a key metabolite required for DNA biosynthesis. Because of DHFR's central location in metabolism and required activity for cell proliferation, it has become an attractive drug target for the treatment of human cancers and infectious diseases. Furthermore, differences in sequence and structural properties in DHFRs from different organisms allow many of these drugs to act with high specificity. Under a separate light, extensive biochemical and structural studies on E. coli DHFR have led to DHFR becoming a paradigm for how dynamic fluctuations in protein structure facilitate enzyme function. DHFR is now known to undergo switching between distinct conformational states on a microsecond to millisecond timescale, in a manner that connects one step in the functional cycle to adjacent steps. Here, E. coli DHFR is studied in light of its importance as a model for both protein-drug interactions and enzyme dynamics. An interdisciplinary experimental approach combining protein NMR relaxation with transient and pre-steady-state kinetics will be employed to study the response of DHFR behavior to chemical denaturants and antifolate inhibitors with affinities spanning five orders of magnitude. Since off-rates are a key determinant of binding affinity, attention will be paid to the role of internal dynamics and conformational changes to ligand dissociation, in the cases of both natural substrates and antifolates. The role of picosecond-nanosecond fluctuations in stabilizing bound ternary states and promoting concerted conformational changes will be assessed, with particular focus on side-chain mobility. The DHFR system presents an excellent opportunity to study the influence of internal dynamics on folate/antifolate ejection from different conformational states; conversely, these studies will address how conformational context defines the dynamics of ligand occupancy and release. Given that conformational changes are collective motions, the inherent connectivity between residues will be mapped using an NMR perturbation-response approach. Throughout this research, emphasis will be placed on DHFR complexes containing reduced nicotinamide adenine dinucleotide phosphate cofactor (NADPH). In summary, this application seeks to gain mechanistic insights into the role of internal dynamics in protein-drug interactions, slow conformational changes, ligand ejection, and intramolecular communication in DHFR. Project Narrative Dihydrofolate reductase is the target for drugs used to treat cancer and infectious diseases, and it serves as a model for understanding protein-drug interactions. By using protein NMR spectroscopy and enzyme kinetics to identify mechanisms of protein flexibility that either stabilize or destabilize drug occupancy, a greater understanding of the determinants of drug binding affinity will be obtained. This new knowledge will increase the efficiency of the design of small molecule inhibitors to dihydrofolate reductase and other proteins.